1. Field of the Invention
The present invention relates to a proteome analysis (global analysis of protein) using a stable isotope.
2. Disclosure of the Related Art
In the field of proteome analysis (global analysis of protein), a PMF (Peptide Mass Finger Printing) analysis method in which a two-dimensional gel electrophoresi's and a mass spectrometer are combined is commonly used. As a next-generation proteome analysis method which will be an alternative to the PMF, for example, approaches using stable isotopes as disclosed in: Steven P. Gygi, Beate Rist, Scott A. Gerber, Frantisek Turecek, Michael H. Gelb and Ruedi Aebersold, Quantitative analysis of complex protein mixtures using isotope-coded affinity tags, Nature Biotechnology, 994-999, 17, 1999; Kirk C. Hansen, Gerold Schmitt-Ulms, Robert J. Chalkley, Jan Hirsch, Michael A. Baldwin and A. L. Burlingame, Mass Spectrometric Analysis of Protein Mixtures at Low Levels Using Cleavable 13C-Isotope-coded Affinity Tag and Multidimensional Chromatography, Molecular & Cellular PROTEOMICS, 299-314, 2, 2003; and, Salvatore Sechi and Yoshiya Oda, Quantitative proteomics using mass spectrometry, Current Opinion in Chemical Biology, 70-77, 7, 2003 have been contrived.
In Hiroki Kuyama, Makoto Watanabe, Chikako Toda, Eiji Ando, Koichi Tanaka and Osamu Nishimura, An Approach to Quantitative Proteome Analysis by Labeling Tryptophan Residues, Rapid Communications in Mass Spectrometry, 1642-1650, 17, 2003, and the international publication WO 2004/002950 pamphlet, a method developed by the present inventors (NBS method) is disclosed. The NBS method uses stable isotope-labeled 2-nitrobenzenesulfenyl chloride (NBSCl) (2-nitro [13C6] benzenesulfenyl chloride) and unlabeled NBSCl (2-nitro [12C6] benzenesulfenyl chloride). Specifically, the method includes the steps of: (1) preparing two states of protein samples, a protein sample I to be analyzed and its reference protein sample II; (2) modifying the protein sample I with either one of 2-nitro [13C6] benzenesulfenyl chloride and 2-nitro [12 C6] benzenesulfenyl chloride, while modifying the protein sample II with the other one of 2-nitro [13C6] benzenesulfenyl chloride and 2-nitro [12C6] benzenesulfenyl chloride; (3) mixing the modified protein sample I and the modified protein sample II with each other; (4) subjecting the resultant mixture of modified proteins to reduction and alkylation followed by digestion into a peptide mixture containing modified peptide fragments and unmodified peptide fragments; (5) enriching/separating the modified peptide fragments from the peptide mixture by using a hydrophobic chromatography column; and (6) conducting mass spectrometry.
One exemplary protocol of NBS method will be described below.                Solubilize two series of protein samples, a test sample and a control sample, respectively, in a solution containing 0.1 w/v % SDS, and denature by heating (100° C., 3 min.).        Add an acetic acid solution dissolving NBS (Heavy) reagent to one of the samples, and an acetic acid solution dissolving NBS (Light) reagent to the other of the samples, to cause NBS modification reaction respectively (room temperature, overnight).        Mix both of the samples and remove unreacted reagents using a desalting column (LH-20).        Dry the sample after desalting and resuspend in a solution containing 0.01 w/v % SDS.        Add TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) to cause reductive reaction (37° C., 30 min.).        Add an iodoacetamide solution to cause alkylation reaction (room temperature, 45 min.).        Add trypsin to cause site-specific cleavage (37° C., 16 hours).        Enrich NBS-modified peptides using an enrichment column (LH-20).        Analyze enriched fractions using a mass spectrometer.        